Abstract
Bioethanol’s importance as a renewable energy carrier led to the development of new devices for the high-throughput screening (HTS) of ethanol-producing microorganisms, monitoring ethanol production, and process optimization. This study developed two devices based on measuring CO2 evolution (an equimolar byproduct of microbial ethanol fermentation) to allow for a fast and robust HTS of ethanol-producing microorganisms for industrial purposes. First, a pH-based system for identifying ethanol producers (Ethanol-HTS) was established in a 96-well plate format where CO2 emission is captured by a 3D-printed silicone lid and transferred from the fermentation well to a reagent containing bromothymol blue as a pH indicator. Second, a self-made CO2 flow meter (CFM) was developed as a lab-scale tool for real-time quantification of ethanol production. This CFM contains four chambers to simultaneously apply different fermentation treatments while LCD and serial ports allow fast and easy data transfer. Applying ethanol-HTS with various yeast concentrations and yeast strains displayed different colors, from dark blue to dark and light green, based on the amount of carbonic acid formed. The results of the CFM device revealed a fermentation profile. The curve of CO2 production flow among six replications showed the same pattern in all batches. The comparison of final ethanol concentrations calculated based on CO2 flow by the CFM device with the GC analysis showed 3% difference which is not significant. Data validation of both devices demonstrated their applicability for screening novel bioethanol-producer strains, determining carbohydrate fermentation profiles, and monitoring ethanol production in real time.
Graphical abstract
Similar content being viewed by others
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Abbreviations
- ETOH:
-
Ethanol
- HTS:
-
High throughput screening
- CFM device:
-
CO2 flow meter
- OD:
-
Optical density
References
Tesfaw A, Assefa F (2014) Current trends in bioethanol production by Saccharomyces cerevisiae: substrate, inhibitor reduction, growth variables, coculture, and immobilization. International scholarly research notices
Brinkman M, Levin-Koopman J, Wicke B, Shutes L, Kuiper M, Faaij A et al (2020) The distribution of food security impacts of biofuels, a Ghana case study. Biomass Bioenerg 141:105695
Stambuk BU, Batista AS, De Araujo PS (2000) Kinetics of active sucrose transport in Saccharomyces cerevisiae. J Biosci Bioeng 89(2):212–214
Schöck T, Becker T (2010) Sensor array for the combined analysis of water–sugar–ethanol mixtures in yeast fermentations by ultrasound. Food Control 21(4):362–369
Lievense JC, Lim HC (1982) The growth and dynamics of Saccharomyces cerevisiae. Annual reports on fermentation processes. Elsevier, pp 211–262
Tikka C, Osuru HP, Atluri N, Raghavulu PCV (2013) Isolation and characterization of ethanol tolerant yeast strains. Bioinformation 9(8):421
Koskinen PE, Lay C-H, Beck SR, Tolvanen KE, Kaksonen AH, Örlygsson J et al (2008) Bioprospecting thermophilic microorganisms from Icelandic hot springs for hydrogen and ethanol production. Energy Fuels 22(1):134–140
Raman N, Pothiraj C (2008) Screening of Zymomonas mobilis and Saccharomyces cerevisiae strains for ethanol production from cassava waste. Rasayan J Chem 1:537–541
Laluce C, Tognolli JO, De Oliveira KF, Souza CS, Morais MR (2009) Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability. Appl Microbiol Biotechnol 83(4):627–637
Wang H, Ji B, Ren H, Meng C (2014) The relationship between lysine 4 on histone H 3 methylation levels of alcohol tolerance genes and changes of ethanol tolerance in S accharomyces cerevisiae. Microb Biotechnol 7(4):307–314
Caspeta L, Coronel J, Montes de Oca A, Abarca E, González L, Martínez A (2019) Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae. Biotechnol Bioeng 116(10):2587–2597
Xin Y, Yang M, Yin H, Yang J (2020) Improvement of ethanol tolerance by inactive protoplast fusion in Saccharomyces cerevisiae. BioMed Res Int. https://doi.org/10.1155/2020/1979318
Karimi K, Tabatabaei M, Sárvári Horváth I, Kumar R (2015) Recent trends in acetone, butanol, and ethanol (ABE) production. Biofuel Res J 2(4):301–308
Vees CA, Neuendorf CS, Pflügl S (2020) Towards continuous industrial bioprocessing with solventogenic and acetogenic clostridia: challenges, progress and perspectives. J Ind Microbiol Biotechnol: Off J Soc Ind Microbiol Biotechnol 47(9–10):753–787
Cornell NW, Veech RL (1983) Enzymatic measurement of ethanol or NAD in acid extracts of biological samples. Anal Biochem 132(2):418–423
Archer M, De Vos B-J, Visser MS (2007) The preparation, assay and certification of aqueous ethanol reference solutions. Accred Qual Assur 12(3):188–193
Seo H-B, Kim H-J, Lee O-K, Ha J-H, Lee H-Y, Jung K-H (2009) Measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process. J Ind Microbiol Biotechnol 36(2):285–292
Miah R, Siddiqa A, Tuli JF, Barman NK, Dey SK, Adnan N et al (2017) Inexpensive procedure for measurement of ethanol: application to bioethanol production process. Adv Microbiol 7(11):743–748
Hessami MJ, Cheng SF, Ambati RR, Yin YH, Phang SM (2019) Bioethanol production from agarophyte red seaweed, Gelidium elegans, using a novel sample preparation method for analysing bioethanol content by gas chromatography. 3 Biotech 9(1):1–8
de Souza Schneider RdC, Junior CS, Fornasier F, de Souza D, Corbellini VA (2018) Bioethanol production from broken rice grains. Interciencia 43(12):846–851
Udugama IA, Gargalo CL, Yamashita Y, Taube MA, Palazoglu A, Young BR et al (2020) The role of big data in industrial (bio) chemical process operations. Ind Eng Chem Res 59(34):15283–15297
Cabaneros Lopez P, Udugama IA, Thomsen ST, Roslander C, Junicke H, Iglesias MM et al (2021) Transforming data to information: a parallel hybrid model for real-time state estimation in lignocellulosic ethanol fermentation. Biotechnol Bioeng 118(2):579–591
Cañete-Carmona E, Gallego-Martínez J-J, Martín C, Brox M, Luna-Rodríguez J-J, Moreno J (2020) A Low-cost IoT device to monitor in real-time wine alcoholic fermentation evolution through CO 2 emissions. IEEE Sens J 20(12):6692–6700
Henning B, Rautenberg J (2006) Process monitoring using ultrasonic sensor systems. Ultrasonics 44:e1395–e1399
Bowler A, Escrig J, Pound M, Watson N (2021) Predicting alcohol concentration during beer fermentation using ultrasonic measurements and machine learning. Fermentation 7(1):34
Veale EL, Irudayaraj J, Demirci A (2007) An on-line approach to monitor ethanol fermentation using FTIR spectroscopy. Biotechnol Prog 23(2):494–500
Gyalai-Korpos M, Fehér A, Barta Z, Réczey K (2014) Evaluation of an online fermentation monitoring system. Acta Aliment 43(1):76–87
Veiga M, Soto M, Méndez R, Lema J (1990) A new device for measurement and control of gas production by bench scale anaerobic digesters. Water Res 24(12):1551–1554
Macias M, Pérez M, Caro I, Cantero D (1995) Automatic gas meter for laboratory fermenters. Biotechnol Tech 9:655–658
Jahreis K, Bentler L, Bockmann Jr, Hans S, Meyer A, Siepelmeyer Jr, et al. (2002) Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132. Journal of bacteriology. 184(19): 5307-16
Bergey DH (1994) Bergey’s manual of determinative bacteriology. Lippincott Williams & Wilkins
Tenny KM, Cooper JS (2017) Ideal Gas Behavior
Benítez T, del Castillo L, Aguilera A, Conde J, Cerdáolmedo E (1983) Selection of wine yeasts for growth and fermentation in the presence of ethanol and sucrose. Appl Environ Microbiol 45(5):1429–1436
Salmon J (1989) Effect of sugar transport inactivation in Saccharomyces cerevisiae on sluggish and stuck enological fermentations. Appl Environ Microbiol 55(4):953–958
Brice C, Sanchez I, Tesnière C, Blondin B (2014) Assessing the mechanisms responsible for differences between nitrogen requirements of Saccharomyces cerevisiae wine yeasts in alcoholic fermentation. Appl Environ Microbiol 80(4):1330–1339
Kerr RA (1998) The next oil crisis looms large–and perhaps close. Am Assoc Adv Sci. https://doi.org/10.1126/science.281.5380.1128
Mielenz JR (2001) Ethanol production from biomass: technology and commercialization status. Curr Opin Microbiol 4(3):324–329
Benton TG, Froggatt A, Wellesley L, Schröder P (2022) The Ukraine war and threats to food and energy security
Sharma N, Sharma N (2021) Screening and molecular identification of hypercellulase and xylanase-producing microorganisms for bioethanol production. Curr Sci 120(5):841
Luo Z, Zeng W, Du G, Liu S, Fang F, Zhou J et al (2017) A high-throughput screening procedure for enhancing pyruvate production in Candida glabrata by random mutagenesis. Bioprocess Biosyst Eng 40(5):693–701
Zeng W, Guo L, Xu S, Chen J, Zhou J (2020) High-throughput screening technology in industrial biotechnology. Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2020.01.001
Lin Y, Chen Y, Li Q, Tian X, Chu J (2019) Rational high-throughput screening system for high sophorolipids production in Candida bombicola by co-utilizing glycerol and glucose capacity. Bioresour Bioprocess 6(1):17
Abalde-Cela S, Gould A, Liu X, Kazamia E, Smith AG, Abell C (2015) High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform. J R Soc Interface 12(106):20150216
Zeng W, Du G, Chen J, Li J, Zhou J (2015) A high-throughput screening procedure for enhancing α-ketoglutaric acid production in Yarrowia lipolytica by random mutagenesis. Process Biochem 50(10):1516–1522
Oter O, Ertekin K, Topkaya D, Alp S (2006) Room temperature ionic liquids as optical sensor matrix materials for gaseous and dissolved CO2. Sens Actuators, B Chem 117(1):295–301
Iwasaka M, Kurita S, Owada N (2012) Properties of bubbled gases transportation in a bromothymol blue aqueous solution under gradient magnetic fields. J Appl Phys 111(7):07B326
Umeh S, Agwuna L, Okafor U (2017) Yeasts from local sources: an alternative to the conventional brewer‟ s yeast. Int J Biotechnol Food Sci 30:191–195
Perfetto R, Del Prete S, Vullo D, Sansone G, Barone C, Rossi M et al (2017) Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the mediterranean mussel, mytilus galloprovincialis. J Enzyme Inhib Med Chem 32(1):632–639
Wood JA, Orr VC, Luque L, Nagendra V, Berruti F, Rehmann L (2015) High-throughput screening of inhibitory compounds on growth and ethanol production of Saccharomyces cerevisiae. BioEnerg Res 8:423–430
El-Dalatony MM, Salama E-S, Kurade MB, Kim K-Y, Govindwar SP, Kim JR et al (2019) Whole conversion of microalgal biomass into biofuels through successive high-throughput fermentation. Chem Eng J 360:797–805
Schalk R, Braun F, Frank R, Rädle M, Gretz N, Methner F-J et al (2017) Non-contact Raman spectroscopy for in-line monitoring of glucose and ethanol during yeast fermentations. Bioprocess Biosyst Eng 40(10):1519–1527
Menevseoglu A, Aykas DP, Hatta-Sakoda B, Toledo-Herrera VH, Rodriguez-Saona LE (2021) Non-invasive monitoring of ethanol and methanol levels in grape-derived pisco distillate by vibrational spectroscopy. Sensors 21(18):6278
Van Dijken JP, Van Den Bosch E, Hermans JJ, De Miranda LR, Scheffers WA (1986) Alcoholic fermentation by ‘non-fermentative’yeasts. Yeast 2(2):123–127
Komatsuzaki N, Okumura R, Sakurai M, Ueki Y, Shima J (2016) Characteristics of Saccharomyces cerevisiae isolated from fruits and humus: Their suitability for bread making
Acknowledgements
The authors are grateful for all support received from ACECR- Mashhad branch.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Gord Noshahri, N., Sharifi, A., Seyedabadi, M. et al. Development of two devices for high-throughput screening of ethanol-producing microorganisms by real-time CO2 production monitoring. Bioprocess Biosyst Eng 46, 1209–1220 (2023). https://doi.org/10.1007/s00449-023-02892-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-023-02892-3